Brain-targeted nano-carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of drug carriers, and discloses a brain-targeted nano-carrier as well as a preparation method and application thereof. The brain-targeted nano-carrier is prepared from the following main preparation raw materials: PLGA-PEG-NHS (poly (lactic-co-glycolic acid)-PEG-NHS, D-mannosamine and a sulfide donor. The brain-targeted nano-carrier utilizes the dual targeting effect of mannose and sulfide, not only can target the brain, but also effectively improves the permeability of the blood-brain barrier, has the characteristics of high targeting and high transmittance, can be used as a carrier to effectively deliver a therapeutic drug to the brain to play a therapeutic effect, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and in particular relates to a brain-targeted nanocarrier and a preparation method and application thereof. Background Art

[0002] In recent years, the incidence of neurological diseases caused by stressful life has been increasing. Clinical and basic research have tried to use nanobiomaterials to deliver drugs for neurological diseases in order to improve the therapeutic effect of drugs. In medicine, nanobiomaterials can not only be used as bionic organs and applied to the human body, but also as drug carriers for drug delivery. As drug carriers, nanobiomaterials have the characteristics of high safety and long circulation, and can be modified with different ligands to give nanobiomaterials specific targeting. In terms of economic benefits, nanobiomaterials have simple components, are easy to prepare, and have stable properties, and can be mass-produced. Although nanobiomaterials have excellent characteristics such as good biocompatibility and easy modification, the obstacle of crossing the blood-brain barrier is the first problem to be solved.

[0003] The blood-brain barrier (BBB) ​​is a special physiological structure composed of vascular endothelial cells, basement membrane and neuronal cells. As an important barrier to protect the brain, it can resist the invasion of foreign substances and protect the central nervous system from harmful substances in the blood. The existence of the blood-brain barrier greatly blocks the entry of drugs into the brain, limiting the therapeutic effect of drugs.

[0004] For drugs to pass through this barrier, they must meet one of the following conditions: 1. Drugs with a molecular weight of 400-500Da and strong lipophilicity can freely pass through the blood-brain barrier; 2. Use targeted modified nanocarriers to carry drugs to the blood-brain barrier, improve the permeability of the blood-brain barrier, and thus increase the concentration of drugs entering the brain.

[0005] Therefore, the present invention aims to provide a nanocarrier that targets the brain and improves the permeability of the blood-brain barrier for delivering drugs to treat neurological diseases. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a brain-targeted nanocarrier and a preparation method and application thereof. The brain-targeted nanocarrier utilizes the dual targeting effects of mannose and sulfide, which can not only target the brain but also effectively improve the permeability of the blood-brain barrier. It has the characteristics of high targeting and high permeability, and can be used as a carrier to effectively deliver therapeutic drugs to the brain to exert therapeutic effects, and has good application prospects.

[0007] The invention provides a brain-targeted nanocarrier, the main preparation raw materials of which include PLGA-PEG-NHS, D-mannosamine and a sulfide donor.

[0008] The present invention uses PLGA-PEG-NHS as a carrier raw material, and adds D-mannosamine and a sulfide donor to modify it. The prepared nanocarrier can not only deliver therapeutic drugs to the brain by utilizing the targeting effect of mannose, but also release hydrogen sulfide molecules through the carried sulfide donor to open the blood-brain barrier. Through the dual targeting effect of mannose and sulfide, the functions of high targeting and high permeability are achieved, and it can be used as an effective delivery carrier for drugs for treating nervous system diseases.

[0009] Preferably, the PLGA-PEG-NHS is PLGA 5000 -PEG 5000 -NHS.

[0010] Preferably, the sulfide donor is PEG-DTC.

[0011] The present invention also provides a method for preparing the above-mentioned brain-targeted nanocarrier, comprising the following steps:

[0012] (1) adding PLGA-PEG-NHS and D-mannosamine to a buffer solution for dissolution, adjusting the buffer solution to alkaline for reaction, and preparing PLGA-PEG-Mannose;

[0013] (2) adding the PLGA-PEG-Mannose and the sulfide donor to a mixed solution of water and an organic solvent for reaction, and then removing the organic solvent to obtain the brain-targeted nanocarrier.

[0014] Preferably, the buffer solution in step (1) is selected from phosphate buffer.

[0015] Preferably, in step (1), the mass ratio of PLGA-PEG-NHS to D-mannosamine is (15-20):1.

[0016] Preferably, the organic solvent in step (2) is tetrahydrofuran or chloroform.

[0017] Preferably, in step (2), the mass ratio of PLGA-PEG-Mannose to the sulfide donor is (3-8):1.

[0018] Preferably, nitrogen is used to remove the organic solvent in step (2).

[0019] Preferably, ultrasound-assisted reaction is used in step (2).

[0020] The present invention also provides the use of the above-mentioned brain-targeted nanocarrier in the preparation of a product for treating brain diseases. Specifically, the brain disease is a nervous system disease. The brain-targeted nanocarrier proposed in the present invention can target the brain and increase the permeability of the blood-brain barrier, and can achieve targeted brain delivery of therapeutic drugs, and has good application prospects in the preparation of products for treating brain diseases.

[0021] The present invention also provides a product for treating brain diseases, comprising the above-mentioned brain-targeted nanocarrier and a therapeutic drug.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] The present invention creatively discovered that hydrogen sulfide can improve the permeability of the blood-brain barrier and promote the entry of drugs into brain lesions. Based on this, the present invention utilizes the brain-targeting effect of mannose molecules and the function of hydrogen sulfide gas molecules to open the blood-brain barrier to prepare brain-targeted nanocarriers with high targeting and high permeability functions, and its effect has been verified by cell and animal model experiments.

[0024] Using the brain-targeted nanocarrier as a delivery vehicle for brain therapeutic drugs can effectively promote the entry of therapeutic drugs into the brain to exert therapeutic effects, providing new ideas and new solutions for the treatment of mental illnesses and brain tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The construction of the in vitro blood-brain barrier model and the permeability experimental results in Example 2; A is the resistance diagram of b.End3 cells monitored for 7 days; B is the fluorescence intensity diagram of PP@P18, PPM@P18, and S-PPM@P18 passing through b.End3 cells at different time points.

[0026] Figure 2 The results of in vivo imaging and organ distribution of healthy mice in Example 3; A is the in vivo imaging image taken at different time points after intravenous administration of PP@P18, PPM@P18, and S-PPM@P18; B is a statistical fluorescence intensity image of PP@P18, PPM@P18, and S-PPM@P18 in the mouse brain; C is the in vivo imaging image of the isolated brain and organs of PP@P18, PPM@P18, and S-PPM@P18 at 4 hours after administration;

[0027] Figure 3 This is a laser confocal image of a mouse brain tissue slice in Example 3, blue is DAPI, and red is nanomaterial;

[0028] Figure 4The results of constructing the PTSD mouse model in Example 4; A is the movement trajectory of the mouse in the last 5 minutes; B is the statistical chart of the percentage of immobility time in the control group and the PTSD model group, ***P<0.001.

[0029] Figure 5 The figures are the results of in vivo imaging and organ distribution of PTSD model mice in Example 4; A is the in vivo imaging image taken at different time points after intravenous administration of PPM@P18 and S-PPM@P18; B is the in vivo imaging image of the isolated brain and organs of PPM@P18 and S-PPM@P18 taken 4 hours after administration; C is the statistical graph of fluorescence mean values ​​at different time points. DETAILED DESCRIPTION

[0030] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0031] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0032] Example 1: Construction of Nanomicelles PP@P18, PPM@P18, and S-PPM@P18

[0033] Using P18 as a simulated drug delivery, the following nanomicelles were prepared:

[0034] (1)PP@P18

[0035] a. Prepare 4 ml of deionized water in a glass bottle, add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0036] b. PLGA 5000 -PEG 5000 -NHS and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1, and tetrahydrofuran was added to 200 μL to obtain a mixed solution 2.

[0037] c. Under ultrasound, the mixed solution 2 was added to the mixed solution 1. After ultrasound for 2 minutes, the organic solvent was removed with nitrogen to obtain nanomicelles PP@P18.

[0038] (2) PPM@P18

[0039] a. 50 mg PLGA 5000 -PEG 5000-NHS was dissolved in a phosphate buffer at pH 7.4, and then 3 mg of D-Mannosamine was added. Ultrasound was used to promote dissolution. The pH value was adjusted to about 8 with triethylamine, and the reaction was allowed to proceed overnight. The reaction solution was collected and dialyzed to remove excess D-Mannosamine. Lyophilization was performed to obtain PLGA 5000 -PEG 5000 -Mannose white powder.

[0040] b. Prepare 4 ml of deionized water in a glass bottle, add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0041] c. PLGA 5000 -PEG 5000 -Mannose and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1, and tetrahydrofuran was added to 200 μL to obtain a mixed solution 2.

[0042] d. Under ultrasound, the mixed solution 2 was added to the mixed solution 1. After ultrasound for 2 min, the organic solvent was removed with nitrogen to obtain the nanomicelles PPM@P18.

[0043] (3)S-PPM@P18

[0044] a. 50 mg PLGA 5000 -PEG 5000 -NHS was dissolved in a phosphate buffer solution at pH 7.4, and then 3 mg of D-Mannosamine was added. Ultrasound was used to promote dissolution. Triethylamine was used to adjust the pH to about 8, and the reaction was allowed to proceed overnight. The reaction solution was collected and dialyzed to remove excess D-Mannosamine. Lyophilization was performed to obtain PLGA 5000 -PEG 5000 -Mannose white powder.

[0045] b. Prepare 4 ml of deionized water in a glass bottle, add 400 μL of tetrahydrofuran (THF) to obtain a mixed solution 1.

[0046] c. PLGA 5000 -PEG 5000 -Mannose, PEG-DTC and P18 were dissolved in tetrahydrofuran at a mass ratio of 5:1:1, mixed well, and tetrahydrofuran was continuously added to 200 μL to obtain a mixed solution 2.

[0047] d. Under ultrasound, the mixed solution 2 was added to the mixed solution 1. After ultrasound for 2 min, the organic solvent was removed with nitrogen to obtain the nanomicelles S-PPM@P18.

[0048] Among them, PLGA 5000-PEG 5000 -NHS was purchased from Shanghai Maokang Biotechnology Co., Ltd. (model MS5903), P18 was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (model S63823), and PEG-DTC was prepared using the relevant method in the article published in Angewandte Chemie International Edition (A Metal-Phenolic Nanosensitizer Performs Hydrogen Sulfide Reprogrammed Oxygen Metabolism for Cancer Radiotherapy Intensification and Immunogenicity).

[0049] Example 2: Construction of in vitro blood-brain barrier model and permeability experiment

[0050] Mouse brain microvascular endothelial cells bEnd.3 were selected as an in vitro blood-brain barrier model, with 2×10 5 The cells were inoculated in a 12-well Transwell chamber with a pore size of 0.4 μm. The volume of the upper chamber was 500 μL and that of the lower chamber was 1 mL. The cells were incubated overnight at 37°C and 5% carbon dioxide. Fresh culture medium was replaced every other day. The resistance of bEnd.3 was tested using a resistance meter on the second day. When the resistance value reached a relatively steady state, the in vitro blood-brain barrier model was considered to be successfully constructed.

[0051] After the in vitro blood-brain barrier model was successfully constructed, drug administration began; 50 μg / mL of PP@P18, 50 μg / mL of PPM@P18, and 50 μg / mL of S-PPM@P18 were added to the Transwell chamber, and fresh culture medium was added to the lower layer; the fluorescence intensity of the lower layer was detected by a fluorescence spectrophotometer at 2, 4, and 8 hours, respectively.

[0052] The experimental results are as follows Figure 1 As shown, Figure 1 Middle A shows that the in vitro blood-brain barrier model has been successfully constructed; Figure 1 Figure B shows that when a fluorescence spectrophotometer was used to monitor the fluorescence intensity of the lower layer P18, the three groups of PP@P18, PPM@P18, and S-PPM@P18 all had a time-dependent ability to penetrate the blood-brain barrier; compared with other groups, S-PPM@P18 showed a significant ability to improve the blood-brain barrier.

[0053] Example 3: Permeability test of healthy mice through the blood-brain barrier

[0054] 5-week-old healthy male C57 mice were selected as animal models, brain hair was removed, and 100μg PP@P18, 100μg PPM@P18, and 100μg S-PPM@P18 were administered through the tail vein, and in vivo imaging observations of small animals were taken at 2, 4, 6, 8, 12, 24, 48, and 72 hours. After the time series shooting was completed, the organs were taken to observe the distribution of organs in the body, and the brain tissue was fixed with 4% paraformaldehyde, gradient dehydrated, and frozen and sectioned, and the fluorescence distribution of brain sections was observed using laser confocal microscopy.

[0055] The results of in vivo imaging and organ distribution are as follows Figure 2 shown. Figure 2 Figures A and B show that S-PPM@P18 group began to accumulate in the brain 30 minutes after administration and reached the highest level at 4 hours. At 4 hours, the brain and organs were removed to take fluorescence images. Figure 2 As shown in Figure C, both PPM@P18 and S-PPM@P18 groups can reach the brain, while S-PPM@P18 group shows stronger penetration. Figure 3 As shown, the results are consistent with the in vivo imaging results.

[0056] Example 4: Construction of animal model of traumatic stress disorder (PTSD) and permeability experiment

[0057] Eight-week-old male C57 mice were first raised in an animal room for a week to familiarize themselves with the environment and reduce stress. The mice were then subjected to electric shock training (placed in an electric shock box for 5 minutes to adapt) and two electric shocks (a 10-minute program was set, with the first 5 minutes being the adaptation phase without electricity; the last 5 minutes being the stimulation phase, with a current intensity of 0.8 mA, electricity was applied once every 10 seconds, each time for 10 seconds, for a total of 15 times). After the electric shock, a scene fear experiment was conducted on the fifth day, and a successful PTSD animal model was selected.

[0058] like Figure 4 As shown, the immobility time of mice in the model group was significantly increased, which was statistically different from that in the control group, indicating that the PTSD mouse model was successfully established.

[0059] The brain hair of the successfully constructed PTSD model mice was shaved, and 100μg of PP@P18, 100μg of PPM@P18, and 100μg of S-PPM@P18 were administered through the tail vein, and the animals were imaged for in vivo observation at 2, 4, 6, 8, 12, 24, 48, and 72 hours. After the time series imaging was completed, the organs were taken and photographed to observe the distribution of organs in the body.

[0060] The results of in vivo imaging and organ distribution are as follows Figure 5As shown, both S-PPM@P18 and PPM@P18 can reach the brain, and S-PPM@P18 shows a stronger advantage in the first 6 hours. Compared with the healthy mice in Example 3, S-PPM@P18 has a longer retention time in PTSD model mice, which can effectively increase the half-life of the drug and prolong the therapeutic effect.

[0061] The embodiments of the present application are described in detail above in conjunction with the accompanying drawings, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present application.

Claims

1. A brain-targeting nanocarrier, characterized in that: Main raw materials for preparing the brain-targeted nanocarrier include PLGA-PEG-NHS, D-mannosamine and a sulfide donor.

2. The brain-targeting nanocarrier according to claim 1, characterized in that: The PLGA-PEG-NHS is PLGA 5000 -PEG 5000 -NHS.

3. The brain-targeting nanocarrier according to claim 1, characterized in that: The sulfide donor is PEG-DTC.

4. The method for preparing the brain-targeted nanocarrier according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) adding PLGA-PEG-NHS and D-mannosamine to a buffer solution for dissolution, adjusting the buffer solution to alkaline for reaction, and preparing PLGA-PEG-Mannose; (2) adding the PLGA-PEG-Mannose and the sulfide donor to a mixed solution of water and an organic solvent to react, and then removing the organic solvent to obtain the brain-targeted nanocarrier.

5. The preparation method according to claim 4, characterized in that: The buffer solution in step (1) is selected from phosphate buffer.

6. The preparation method according to claim 4, characterized in that: The mass ratio of PLGA-PEG-NHS to D-mannosamine in step (1) is (15-20):

1.

7. The preparation method according to claim 4, characterized in that: The organic solvent in step (2) is tetrahydrofuran or chloroform.

8. The preparation method according to claim 4, characterized in that: In the step (2), the mass ratio of PLGA-PEG-Mannose to the sulfide donor is (3-8):

1.

9. Use of the brain-targeted nanocarrier according to any one of claims 1 to 3 in preparing a product for treating brain diseases.

10. A product for treating brain diseases, characterized in that: The invention comprises the brain-targeted nanocarrier according to any one of claims 1 to 3 and a therapeutic drug.

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